Interferons (IFNs) are a family of small molecule proteins or glycoproteins produced by eukaryotic cells in response to viral infection and other antigenic stimuli, which display broad-spectrum antiviral, antiproliferative and immunomodulatory effects. IFNs have been widely applied in the treatment of various conditions and diseases, such as viral infections, e.g. hepatitis B, hepatitis C and HIV; inflammatory disorders and diseases, e.g. multiple sclerosis, arthritis, asthma, cystic fibrosis and interstitial lung disease; and tumors, e.g. myelomas, lymphomas, liver cancer, lung cancer, hairy-cell leukemia, and so on (Kenji Oritani, Paul W Kincade, et al. Type I interferon and limitin: a comparison of structures, receptors, and functions. Cytokine and Growth Factor Reviews, 12, 337-348, 2001; Yu-Sen Wang, Stephen Youngster, et al. Structural and biological characterization of PEGylated recombinant interferon alpha-2b and its therapeutic implications. Advance Drug Delivery Reviews, 54, 547-570, 2002).
IFNs are classified into four types according to their differences in chemical, immunological, and biological properties: interferon-α, β, γ and ε. Interferon-α (IFN-α) is secreted by leukocytes. Human IFNs-α are encoded by a multigene family consisting of about 20 genes, the encoded proteins sharing up to about 90% amino acid sequence homology (Henco K., Brosius F. J., et al. J. Mol. Biol., 185, 227-260, 1985). Human IFN-α2b is one of the subtypes of the α2 subfamily of human IFN-α family, and is a single chain protein with various biological activities. The single chain protein consists of 165 amino acid residues with 4 Cys, wherein two intrachain disulfide bonds are formed between Cys1-Cys98 and Cys29-Cys138 respectively and the N-terminal amino acid is Cys with one free α-NH2 group. The residues in positions 31, 49, 70, 83, 112, 121, 131, 133, 134 and 164 of the amino acid sequence are Lys, each of which contains one free ε-NH2 group. This protein is not glycosylated and sensitive to many proteases. The amino acid sequence of human interferon-α2b is shown in SEQ ID No: 1.
IFNs are usually administered parenterally in clinical treatments. The short in vivo half-life (2-4 h) and strong immunogenicity of IFNs result in a shorter dosing interval and a higher dosing frequency. As the generated antibodies significantly decrease the therapeutic efficacy, it is difficult to achieve ideal clinical efficacy. The polyethylene glycol (PEG) modification technology developed in recent years has provided a possible solution to the above problems.
PEG is an inert, nontoxic and biodegradable organic polymer, and is important in the fields of both biotechnology and pharmaceutics. PEG modification technique is to link PEG to an active protein via covalent bond. After the polyethylene-glycolation (PEGylation), the properties of the protein can be significantly improved, e.g. the prolongation of drug metabolic half-life, the reduction of immunogenicity, the increase of safety, the improvement of therapeutic efficacy, the decrease of dosing frequency, the increase of drug solubility/water solubility, the increase of resistance against proteolysis, the facilitation of drug controlled release and so on. For further details please refer to Inada et al. J. Bioact. and Compatible Polymers, 5, 343, 1990, Delgado et al. Critical Reviews in Therapeutic Drug Carrier Systems, 9, 249, 1992, Katre. Advanced Drug Delivery Systems, 10, 91, 1993, and U.S. Pat. No. 4,179,337.
It is disclosed in U.S. Pat. No. 4,179,337, after linking PEG to an enzyme or insulin, the immunogenicity of the protein was reduced, while simultaneously the activities of the protein were reduced as well. This was also found in G-CSF (Satake-Ishikawa et al. Cell Structure and Function, 17, 157-160, 1992), IL-2 (Katre et al. Proc. Natl. Acad. Sci. USA, 84, 1487, 1987), TNF-α (Tsutsumi et al. Jpn. J. Cancer Res., 85, 9, 1994), IL-6 (Inoue et al. J. Lab. Clin. Med., 124, 529, 1994) and CD4-IgG (Chamow et al. Bioconj. Chem., 5, 133, 1994).
It was reported that the branched PEG-modified protein exhibited better pH tolerance, thermo-stability and resistance against proteolysis than linear chain PEG-modified proteins (Monfardini et al. Bioconjugate Chem., 6, 62, 1995). Generally, a PEG molecule modifies a protein by linking itself to the N-terminal α-amino group or ε-amino groups of a Lys residue within the protein molecule. There are normally three types of PEGs for protein modification: a linear chain PEG (EP 0593868), an U-shaped branched PEG (EP 0809996) and an Y-shaped branched PEG (CN1243779C).
Currently many kinds of PEGylated proteins have been applied clinically. In 1990, the PEGylated-bovine adenosine deaminase (Adagen) produced by ENZON Inc. was approved by FDA, and used to treat severe combined immunodeficiency disease (pegfamg013102LB). In 1994, another PEG-modified protein for treating acute lymphoblastic leukemia, the PEGylated asparaginase (pegaspargase, Oncaspar), was also marketed in US (103411s5052lbl). The PEG modified interferon-α2b (PEG IFN-α2b, PEG-Intron) developed by Schering-Plough was approved by FDA for marketing in 2000 and the PEGylated interferon-α (PEG IFN-α2a, Pegasys) produced by Hoffman-la Roche Ltd. was also approved for marketing in 2002, both of which are used to treat hepatitis (103964s5037lbl, pegsche011901LB). In 2002, the PEG modified human granulocyte colony-stimulating factor produced by Amgen Inc. (PEG-filgrastim, Neulasta) was also approved by FDA, which is used to treat metastatic breast cancer (pegfamg013102LB). The FDA also accepted the application for PEGylated human growth factor antagonist developed by Pharmacia. The PEG combined TNF-α antibody fragment from Celltech and the PEG-TNF receptor from Amgen are tested in the advanced clinical trials. The first PEG-organic molecule conjugate, PEGylated camptothecin, has also entered phase II of clinical trial. In 2004, the PEG modified oligonucleotide (Pegaptanib, Macugen™) was approved by FDA. The in vivo metabolism of the PEG in the drug (or PEG itself) has already been clearly understood, and PEG has been proven to be a good and safe drug modifier without any adverse effect.
The PEGs that can be linked to a protein drug normally need derivatization, so that one or two terminal groups of the ends of PEGs can be chemically activated to possess a proper functional group which displays activity, and thus can form a stable covalent bond with, at least one functional group of the drug to be linked. For example, PEGs can be linked to ε-NH2 of Lys residue within the protein peptide chain, or to α-NH2 of the N-terminal amino acid residue of the protein peptide chain. In the PEGylation of IFN-α described in European patent EP0809996, PEG-NHS is linked through nucleophilic substitution to α-NH2 of the N-terminal amino acid or ε-NH2 of Lys in IFN-α. The PEG-NHS mentioned in the above patent is a U-shaped branched PEG derivative (PEG2-NHS), the molecular formula thereof as below:
wherein, R and R′ are independently a low molecular weight alkyl group, n and n′ are from 600 to 1500, and the average molecular weight of the PEG is from 26 KD to 66 KD. The molecular formula of the PEG2-NHS-modified IFN-α is as below:

Where one or more PEG2-NHS molecules are linked to α-NH2 of the N-terminal amino acid or ε-NH2 of Lys in IFN-α, the obtained products are a mixture of non-PEGylated IFNs-α, PEGylated IFNs-α at a single amino acid residue and PEGylated IFNs-α at multiple amino acid residues. The PEGylated IFN-α at a single amino acid residue can be isolated from the obtained products by any appropriate purification means. IFN-α has one N-terminal amino acid and more than one Lys residues, namely several reactive sites for PEG2-NHS, so the isolated PEGylated IFNs-α at a single amino acid residue are a mixture of the isomers of the PEGylated IFNs-α at different single amino acid residues.
In European patent EP 0593868, linear-chain PEG is used to modify IFN, the molecular formula of the modified product as below:
wherein R is a low molecular weight alkyl group; R1, R2, R3 and R4 are H or low molecular weight alkyl groups; m is from 1 to the number of possible PEG modification positions in IFN; W is O or NH; x is from 1 to 1000, y and z are from 0 to 1000, x+y+z is from 3 to 1000; and at least one of R1, R2, R3 and R4 is a low molecular weight alkyl group. Yu-Sen Wang et al (Yu-Sen Wang et al, Advanced Drug Delivery Reviews, 54: 547-570, 2002. Yu-Sen Wang et al, Biochemistry, 39, 10634-10640, 2000.) have reported the modification of rIFN-α2b with 12 KD linear monomethoxy-PEG (Peg-Intron) and shown that the products analyzed by HPLC-IE are a mixture of more than 14 isomers modified by PEG at different single amino acid residues. The molecular formula of the linear PEGs used by Yu-Sen Wang et al is shown below:
wherein the average molecular weight of the PEG is 12 KD.